LOCUS       62056_16755        5601 bp DNA     circular SYN 07-JAN-2021
DEFINITION  Cloning vector pMESP23E2, complete sequence.
ACCESSION   MT338521
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5601)
  AUTHORS   Uchanski T, Masiulis S, Fischer B, Kalichuk V, Lopez-Sanchez U, 
            Zarkadas E, Weckener M, Sente A, Ward P, Wohlkoenig A, Zoegg T, 
            Remaut H, Naismith JH, Nury H, Vranken W, Aricescu AR, Pardon E, 
            Steyaert J.
  TITLE     Megabodies expand the nanobody toolkit for protein structure 
            determination by single-particle cryo-EM
  JOURNAL   Nat Methods 18 (1), 60-68 (2021)
REFERENCE   2  (bases 1 to 5601)
  AUTHORS   Steyaert J, Uchanski T, Pardon E.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-APR-2020) VIB-VUB Center for Structural Biology, VIB, 
            Pleinlaan 2, Brussels 1050, Belgium
REFERENCE   3  (bases 1 to 5601)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods"; date: "2021"; volume: "18"; issue: "1"; pages: "60-68"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (14-APR-2020) VIB-VUB Center for Structural Biology, VIB, Pleinlaan 
            2, Brussels 1050, Belgium"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     sig_peptide     225..290
                     /label=pelB signal sequence
                     /note="leader peptide for secretion"
     misc_feature    291..326
                     /label=nanobody beta-strain A (residues 1-13)
                     /note="nanobody beta-strain A (residues 1-13)"
     misc_feature    330..2654
                     /label=cYgjK scaffold protein
                     /note="cYgjK scaffold protein"
     misc_feature    2658..2690
                     /label=multi cloning site
                     /note="multi cloning site"
     CDS             2691..2708
                     /label=6xHis
                     /note="6xHis affinity tag"
     misc_feature    2709..2720
                     /label=EPEA-tag
                     /note="EPEA-tag"
     primer_bind     complement(2730..2746)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      2959..3414
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3696..3800
                     /label=AmpR promoter
     CDS             3801..4658
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4832..5420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agagcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacaagctta aggagacagt acatatgaaa tacctattgc
      241 ctacggcagc cgctggattg ttattactcg cggcccagcc ggccatggcc caggtgcagc
      301 tggtggagtc tgggggagga ctagtatata aagaagagac gcagtctggg ctgaacaact
      361 acgcccgtgt ggtggagaaa ggccagtacg acagtctgga aattcctgca caggttgctg
      421 catcgtggga atcggggcgt gatgatgccg ccgtctttgg gtttatcgac aaagaacagt
      481 tggataaata tgtcgctaat ggcggcaaac gtagcgactg gacagtgaaa ttcgccgaaa
      541 accgtagtca ggacggaaca ttgctgggct actcgctttt gcaggagtcg gtggatcagg
      601 ccagctatat gtacagcgat aaccattatc tggcagagat ggcaaccatc ctgggtaagc
      661 cagaagaggc caaacgttat cgtcagttgg cacagcagct ggcagactac atcaacacct
      721 gtatgttcga ccctactacc cagttctact atgacgtacg tattgaagat aaaccactgg
      781 ctaacggctg cgcaggcaaa cctattgttg agcgtggtaa agggccagaa ggctggtcgc
      841 cactgtttaa cggtgcagca actcaggcca atgccgacgc tgtggtgaag gtgatgcttg
      901 atcctaaaga gttcaacacc tttgtcccac tgggaactgc cgctttaacc aatcctgctt
      961 ttggcgctga catctactgg cgtgggcgtg tatgggtgga tcagttctgg tttggtctga
     1021 aagggatgga gcgttacggt tatcgtgatg atgccctgaa gctggcagat acattcttcc
     1081 ggcacgccaa agggttaacc gccgatggac caattcagga aaattacaac ccactgacag
     1141 gcgcacagca aggcgcacca aatttctcct ggagcgctgc tcatttgtat atgttgtata
     1201 acgatttttt ccgtaagcaa gctagcggtg gaggatcagg aggtggagga tcaggaggtg
     1261 gaggctcagg taacgccgac aattataaaa acgtgattaa ccgtactggg gcccctcagt
     1321 acatgaagga ttacgattac gacgatcacc agcgttttaa tccatttttc gatcttggag
     1381 cctggcatgg tcatctgttg ccagacggcc ctaacactat gggcggcttt ccaggcgttg
     1441 ctctgctgac agaagagtac atcaacttta tggcaagcaa tttcgaccgt ctgaccgtct
     1501 ggcaggacgg caagaaagta gactttacac tggaggcata cagtattcct ggtgctctgg
     1561 tgcaaaaact gacagcaaaa gatgtgcaag tcgaaatgac tctgcgtttc gccactcctc
     1621 gtacctcact tctggaaacc aaaatcacca gcaataaacc actggatctg gtgtgggacg
     1681 gcgaactgct ggaaaaactg gaagctaaag aagggaaacc tctttccgat aaaaccattg
     1741 ctggcgaata ccctgactat cagcgtaaaa tcagcgccac ccgtgatggc ctgaaagtca
     1801 cctttggcaa agtgcgtgcc acctgggatc tgctgacctc cggcgaatcg gaatatcagg
     1861 tgcataaatc cctgcctgtg cagactgaaa tcaacggtaa tcgttttacc agtaaggccc
     1921 atatcaacgg ttcgaccaca ctttacacca cctattccca tctgctgacc gcacaggaag
     1981 ttagcaaaga gcaaatgcag atccgtgata ttctggctcg tccagcattt tatctgaccg
     2041 cctcgcagca acgttgggaa gaatacctga agaaagggtt aaccaatcca gatgcaaccc
     2101 ctgaacagac tcgtgtcgct gtgaaagcaa tcgaaactct gaacggtaac tggcgttcac
     2161 ctggcggtgc tgtgaaattt aacaccgtta caccatcggt gacagggcgt tggttctccg
     2221 gcaatcagac ctggccttgg gatacctgga agcaggcttt tgctatggca catttcaatc
     2281 cagacatcgc caaagagaat atccgtgctg tcttctcctg gcagatccag cctggcgata
     2341 gcgtgcgtcc acaggatgtg ggctttgtcc cagacctgat tgcttggaac ctgagtccag
     2401 agcgtggtgg cgatggcggc aactggaacg aacgtaatac caaacctagc cttgccgcct
     2461 ggtcggtgat ggaagtgtat aacgtcaccc aagataaaac ctgggtggca gagatgtacc
     2521 ctaaactggt ggcctatcac gactggtggt tacgtaaccg tgatcacaac ggcaacggcg
     2581 tgccagaata tggcgcaacc cgtgacaaag cccacaacac tgagagcggc gagatgctgt
     2641 ttacagtgaa aaaggattcc ggaagagctt agtcgacaag ctcttcaagt caccaccatc
     2701 accatcacga acctgaagcc taagaattca ctggccgtcg ttttacaacg tcgtgactgg
     2761 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2821 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2881 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     2941 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3001 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3061 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3121 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     3181 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3241 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3301 gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3361 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     3421 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3481 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3541 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3601 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3661 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3721 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3781 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3841 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3901 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3961 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4021 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4081 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4141 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4201 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4261 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4321 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4381 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4441 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4501 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4561 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4621 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4681 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4741 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4801 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4861 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4921 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4981 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5041 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5101 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5161 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5221 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5281 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5341 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5401 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5461 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5521 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5581 cagtgagcga ggaagcggaa g
//
